What Do We Have to Know about PD-L1 Expression in Prostate Cancer? A Systematic Literature Review (Part 6): Correlation of PD-L1 Expression with the Status of Mismatch Repair System, BRCA, PTEN, and Other Genes

Pembrolizumab (anti-PD-1) is allowed in selected metastatic castration-resistant prostate cancer (PC) patients showing microsatellite instability/mismatch repair system deficiency (MSI-H/dMMR). BRCA1/2 loss-of-function is linked to hereditary PCs and homologous recombination DNA-repair system deficiency: poly-ADP-ribose-polymerase inhibitors can be administered to BRCA-mutated PC patients. Recently, docetaxel-refractory metastatic castration-resistant PC patients with BRCA1/2 or ATM somatic mutations had higher response rates to pembrolizumab. PTEN regulates cell cycle/proliferation/apoptosis through pathways including the AKT/mTOR, which upregulates PD-L1 expression in PC. Our systematic literature review (PRISMA guidelines) investigated the potential correlations between PD-L1 and MMR/MSI/BRCA/PTEN statuses in PC, discussing few other relevant genes. Excluding selection biases, 74/677 (11%) PCs showed dMMR/MSI; 8/67 (12%) of dMMR/MSI cases were PD-L1+. dMMR-PCs included ductal (3%) and acinar (14%) PCs (all cases tested for MSI were acinar-PCs). In total, 15/39 (39%) PCs harbored BRCA1/2 aberrations: limited data are available for PD-L1 expression in these patients. 13/137 (10%) PTEN- PCs were PD-L1+; 10/29 (35%) PD-L1+ PCs showed PTEN negativity. SPOP mutations may increase PD-L1 levels, while the potential correlation between PD-L1 and ERG expression in PC should be clarified. Further research should verify how the efficacy of PD-1 inhibitors in metastatic castration-resistant PCs is related to dMMR/MSI, DNA-damage repair genes defects, or PD-L1 expression.

The PD-1/PD-L1 pathway leads to the inactivation of PD-1 expressing cells, in particular CD8+ cytotoxic T cells, favoring tumor immune escape but also representing a target for immunotherapy drugs [2,3,. Despite some limitations of this approach, PD-L1 immunohistochemical expression is frequently tested in various tumor types to select patients for immunotherapy administration [158][159][160][161][162].
Pembrolizumab (anti-PD-1) is a highly selective IgG4-kappa humanized monoclonal antibody and immune checkpoint inhibitor; it binds with high affinity to the cell surface receptor PD-1 (expressed by T cells), blocking the inhibitory pathway, triggering a physiological shift to immune reactivity, and enhancing the antitumor immune response [4,9,23,26,34,64,156]. Recent evidence revealed good therapeutic activity of this drug in monotherapy, and the 2021 United States National Comprehensive Cancer Network (NCCN) guidelines have allowed pembrolizumab (as a second-line therapy or beyond) for selected patients with metastatic castration-resistant PCs (mCRPCs) showing high microsatellite instability/mismatch repair system protein deficiency (MSI-H/dMMR) or tumor mutation burden (TMB) >10 mutations/Mb [4]. In particular, the MSI/MMR status testing is recommended in mCRPC patients, and it may be considered in men with regional or castration-naïve metastatic PC [4]. For these reasons, in current practice, the International Society of Urologic Pathology recommends, if clinically indicated, MMR assessment via immunohistochemical analysis of MSH2, MSH6, MLH1, and PMS2 protein expression, with or without MSI testing, sequencing of MMR genes, and/or TMB estimate [6]. However, the dMMR/H-MSI status, as well as PD-L1 expression, may not necessarily correlate to the response to targeted immunotherapy, and combined analysis of MMR/MSI and PD-L1 status in PC patients was rarely performed [19,23,38,47,53,61,68,77,92,156].
TMB is a phenotypic hallmark of MSI-H/dMMR tumors [4]. Various genetic mutations, chromosomal aberrations, and molecular changes have been found in the genome of PC; however, most of them are still under investigation [10]. Germline mutations in MMR genes are drivers for the Lynch syndrome, which is an autosomal dominant genetic disorder causing multiple cancers (including PC) in affected patients [4]. In addition, the Breast Cancer 1-2 (BRCA1/2) genes are main regulators of a cellular DNA damage repair system (homologous recombination repair, HRR); the failure of HRR is due to combined germline and somatic mutations of BRCA1/2 and/or other genes, favoring the activation of alternative and less effective DNA repair pathways (such as base/nucleotide excision repair or mismatch repair system) [7][8][9]19,23,53,56,92,163,164]. Indeed, BRCA1/2 mutations are responsible for a group of hereditary PCs; specific drugs (poly-ADP ribose polymerase inhibitors, PARPi) can be administered to BRCA-mutated PC patients, according to the NCCN guidelines [4,9,19,23,53,56,92]. HRR gene testing is recommended for metastatic PC patients and considered for PC with spread to regional lymph nodes [4]. To improve the survival of mCRPC patients, treatment combinations (such as immunotherapy and PARPi) have been increasingly tested; however, few studies simultaneously tested PARPi and drugs affecting the PD-1/PD-L1 pathway; combined analysis of the BRCA and PD-L1 status was rarely conducted in PC patients [7][8][9]19,23,53,56,92].
We have performed a systematic literature review of the studies combining the analysis of PD-L1 expression with the investigation of the MSI/MMR, PTEN, and/or BRCA status in PC, trying to delineate the potential links between these markers in PC patients. The retrieved data concerning other main genes involved in PC genesis and progression are also discussed.
TMB is a phenotypic hallmark of MSI-H/dMMR tumors [4]. Various genetic mutations, chromosomal aberrations, and molecular changes have been found in the genome of PC; however, most of them are still under investigation [10]. Germline mutations in MMR genes are drivers for the Lynch syndrome, which is an autosomal dominant genetic disorder causing multiple cancers (including PC) in affected patients [4]. In addition, the Breast Cancer 1-2 (BRCA1/2) genes are main regulators of a cellular DNA damage repair system (homologous recombination repair, HRR); the failure of HRR is due to combined germline and somatic mutations of BRCA1/2 and/or other genes, favoring the activation of alternative and less effective DNA repair pathways (such as base/nucleotide excision repair or mismatch repair system) [7][8][9]19,23,53,56,92,163,164]. Indeed, BRCA1/2 mutations are responsible for a group of hereditary PCs; specific drugs (poly-ADP ribose polymerase inhibitors, PARPi) can be administered to BRCA-mutated PC patients, according to the NCCN guidelines [4,9,19,23,53,56,92]. HRR gene testing is recommended for metastatic PC patients and considered for PC with spread to regional lymph nodes [4]. To improve the survival of mCRPC patients, treatment combinations (such as immunotherapy and PARPi) have been increasingly tested; however, few studies simultaneously tested PARPi and drugs affecting the PD-1/PD-L1 pathway; combined analysis of the BRCA and PD-L1 status was rarely conducted in PC patients [7][8][9]19,23,53,56,92].
We have performed a systematic literature review of the studies combining the analysis of PD-L1 expression with the investigation of the MSI/MMR, PTEN, and/or BRCA status in PC, trying to delineate the potential links between these markers in PC patients. The retrieved data concerning other main genes involved in PC genesis and progression are also discussed.

Materials and Methods
Our systematic literature review was conducted according to the "Preferred Reporting Items for Systematic Reviews and Meta-Analyses" (PRISMA) guidelines (http://www.prisma-statement.org/; accessed on 8 May 2021) ( Figure 1).  We summarized the literature data concerning the role of PD-L1 in PC, describing the clinic-pathologic features of the published cases, and answering the following "Population, Intervention, Comparison, Outcomes" (PICO) questions: • Population: patients, tumor cell lines, and mouse models included in studies concerning the role of PD-L1 in PC. After duplicates exclusion, two independent reviewers screened the titles and abstracts of the 560 resulting records in order to verify the eligibility, inclusion, and exclusion criteria. The selected articles were obtained in full-text format: two other co-authors verified the relevance of the studies and searched for further pertinent references. Finally, two authors checked the extracted information. Globally, 155 full texts were considered for eligibility and, after reading them, seven articles were excluded as they were unfit according to the inclusion/exclusion criteria or because they presented scant or aggregated data; 148 articles were finally included [8,.
Data collection was study-related (authors and year of study publication) and caserelated (tumor stage at presentation, Grade Group, type of specimen, treatment, test methods, results of PD-L1 expression, follow-up and outcomes, experiment type).
The collected data were described as continuous variables (summarized by ranges, mean, and/or median values) or categorical variables (analyzed by frequencies and percentages).
As there was a lot of information to discuss, we have divided the presentation of our data into different articles, focusing on different sub-topics. Here, we analyze the studies of PC patients simultaneously evaluating PD-L1 expression and at least one of the following: status of mismatch repair system/microsatellite instability, BRCA, or PTEN genes. The potential correlations between PD-L1 expression and some of the other main genes involved in PC genesis and progression are also briefly discussed. Table 1 reports the results of the studies on human PC patients, investigating both PD-L1 expression and the MMR/MSI status; supplementary data are reported in Table S1.
Few interesting results concerning the potential correlation of MMR/MSI with clinical outcome were reported in the studies resulting from our review. Some authors [61] found that dMMR was associated with decreased OS (n = 124; p = 0.005), while others (n = 220) [29,37] described that cases with loss of ≥1 MMR protein and PD-L1 expression in tumor-infiltrating lymphocytes (TILs) had a significantly higher risk of biochemical recurrence (BCR) (p = 0.045). No convincing association was identified as regards Gleason score, stage, age, or other variables. The reported series were usually retrospective, sometimes harboring selection biases and/or testing a few samples. Moreover, limited clinic-pathologic information was available in some studies, while the PD-L1 and MMR/MSI statuses were variably analyzed (different assays or antibody clones; variable scoring systems), and their potential correlation was rarely or unclearly investigated. Finally, studies were typically monocentric: larger validation cohorts from multiple hospitals are required.
None of the six responders to pembrolizuab showed MSI in another series [9]. Moreover, Petrylak et al. [19] analyzed the MSI status of 14 CRPCs: 1 dMMR (MSH2-/MSH6-) CRPC responded to atezolizumab, while another MSI-H PC did not benefit from treatment. However, the PD-L1 status of these cases was unclear, despite all the tested tumors showing absent expression or <5% positivity rate.

PD-L1 and BRCA1/2 Status in PC Patients
Few studies analyzed PD-L1 expression and the status of BRCA1/2 and other DDR genes in human PC patients (Table 2); additional information are reported in Tables 1 and S1.
Combining the scant analyzable data, aberrations in BRCA1/2 genes were found in 15/39 (39%) cases [19,53,56], involving BRCA1 (n = 1), BRCA2 (n = 13), or both genes (n = 1). Moreover, 19/153 (12%) PCs of another series showed BRCA1/2 or ATM mutations [9]. Unfortunately, the relation of PD-L1 expression with the BRCA status was unclear for most of the cases; only 1/10 (10%) PCs with BRCA aberrations was clearly PD-L1+ [19,53]. Some authors [53] found no association (p > 0.05) between the DNA mutational profile, CD3/8 or PD-L1 IHC status, RNA-seq CD8, or TMB, and the expression profile of any analyzed genes, including BRCA1/2. The scant available information did not allow us to discuss the potential differences in PD-L1 expression between PCs with somatic vs. germline BRCA mutations. PD-1 inhibition has activity in mCRPCs, including tumors without MSI, DDR defects, or PD-L1 expression [23,92]. The phase II KEYNOTE-199 study reported that pembrolizumab showed antitumor activity and disease control with acceptable safety in docetaxel-refractory mCRPCs, regardless of the PD-L1 status, in both RECIST-measurable and non-measurable cases: 19/153 (12%) PCs had aberrations in BRCA1/2 or ATM, while 10/153 (7%) PCs revealed alterations in ≥1 of 12 other HRR genes. Patients with somatic mutations in BRCA1/2 or ATM showed higher response rates compared to the HRR-aberrant or HRR-normal groups (11%, 0%, and 3%, respectively). Unfortunately, it was not com-pletely clear if there was a correlation between the BRCA and the PD-L1 status [165]. In another series [19], most of the CRPC patients with DDR genes alterations did not respond to atezolizumab (anti-PD-L1), and the DDR status was not a strong predictor of clinical benefit. Finally, some authors [56] included responders to durvalumab (anti-PD-L1) plus olaparib that harbored germline mutations in DDR genes, including BRCA2 and NBN, but it was unclear if these cases were tested for PD-L1 by IHC.

PD-L1 and PTEN Status in PC Patients
Very few studies performed a combined analysis of PD-L1 expression and PTEN status in human PC patients by using molecular or IHC assays (Table 3) [19,38,53,77,86,96]; additional information is reported in Tables 1, 2 and S1.  The assays and IHC scoring systems of PTEN and PD-L1 were variable and sometimes unclear. It was difficult to correlate the scant results in some cases; when data were analyzable, 179/326 (55%) PCs showed PTEN loss by IHC: 13/137 (10%) of PTEN-cases were PD-L1+, while 10/29 (35%) of PD-L1+ cases showed PTEN negativity [77,86,96]. In the series of Calagua et al. [77], the PD-L1 high and PD-L1 low components of the cases showing a nodular PD-L1 positivity pattern (n = 7; 39%) revealed a consistently concordant ERG status but a variable PTEN staining pattern. Conversely, Shaw et al. found no significant association between PD-L1+ tumors and ERG/PTEN status [38].
In a Phase I trial [35] testing PT-112 (plus or without Avelumab) on CRPCs, a case with PIK3CB mutation and PTEN loss showed a 4-month response (93% PSA decrease, 48% reduction in target lesions); the PD-L1 status was unclear.

PD-L1 and Other Genes Involved in PC Genesis and Progression
The TP53 gene encodes 12 isoforms through alternative promoters, translation start sites, and RNA splicing [42]. In a study [42], increased expression of the ∆133TP53β isoform defined high-risk PC patients, while PCs showing increased ∆40TP53 and TP53α mRNAs levels revealed good prognosis. High ∆133TP53β mRNA levels alone predicted poor outcome (accuracy: 88%), correlating to advanced stage, increased proliferative index, immune cell infiltrate (T cells, macrophages, immunosuppressive cells), PD-1 overexpression on T cells, and PD-L1 upregulation in PC cells and CD163+ CSF1R+ macrophages [42]. Indeed, this isoform increased the expression of PD-L1 mRNA levels-while its knockdown reduced them-and of genes involved in immune signaling and migration. IL-6 was directly regulated by ∆133p53; its transcription may be induced by hypoxia in T53-wild-type tumors [42]. In a large series analyzing The Cancer Genome Atlas (TCGA) database, the immune class subtype of PCs revealed higher TP53 mutation frequency than the nonimmune class sub-group (14% vs. 10%) (especially vs. the immune-suppressed PCs: 19%, p < 0.001) [21]. TP53 mutations were also found in 14 cases of our series [9,19,26,53], but the PD-L1 status of these cases was unclear.
MMR deficiency could be associated with aggressive clinic-pathologic features in PC (high Gleason score, advanced stage, decreased OS); dMMR mutational signatures were sometimes associated with increased T cell-related and checkpoint-related transcripts (including PD-L1 and PD-L2) [61,167,168]. In another series [29,37], loss of ≥1 MMR protein and PD-L1 expression in TILs had a significantly higher BCR risk.
An association between MSI-H status and the efficacy of PD-L1/PD-1 inhibitors has been established in a range of solid tumors, including a relatively small number of PC cases; MMR deficiency was sometimes associated with favorable response to anti-PD-1 therapy or PD-L1 expression [29,37,61,[167][168][169][170][171][172][173][174]. The 2021 NCCN guidelines have allowed immunotherapy in men with asymptomatic or minimally symptomatic mCRPCs; pembrolizumab can be administered as ≥2nd line systemic therapy to dMMR/MSI-H mCRPCs that have progressed through prior docetaxel and/or novel hormone therapy [4]. Durable clinical benefit, sometimes with a 50% reduction in PSA levels, was occasionally reported in PC patients receiving checkpoint inhibitors as monotherapy or combination therapy, although with variable response rates: it was usually unclear if PD-L1 was tested in all these cases [48,167,175]. However, the dMMR/H-MSI status does not necessarily correlate to the response to immunotherapy: emerging clinical data have reported that dMMR/MSI-H mCRPCs do not always respond, while responders are not necessarily dMMR/MSI-H (by IHC and/or polymerase chain reaction analysis) [48,61].
The NCCN guidelines recommend using a metastatic biopsy for histologic and molecular analysis of somatic tumor testing [4]. Indeed, the MSI-H/dMMR phenotype may be acquired somatically during disease evolution; in a study, MSI may have been subclonal in earlier samples of two longitudinally profiled PCs, which is probably due to tumor heterogeneity (not favoring a truncal event) [48]. Unfortunately, the NCCN guidelines [4] do not define how to evaluate the MSI-H/dMMR or the PD-L1 status: MSI-H or dMMR can be tested by using either DNA tests or IHC. The second approach is easy to use and cost-effective, but it may be insufficient to predict PC recurrence after radical prostatectomy [29,38,61,[168][169][170][171][172][173][174]. In case of molecular testing, validated NGS assays are preferred by the NCCN guidelines [4]. NGS can assess for the MSI-H/dMMR status by interrogating microsatellite loci to identify MSI, finding mutations and copy number alterations in MMR-associated genes; it can also infer the TMB, which is a phenotypic hallmark of MSI-H/dMMR tumors [176,177]. In clinical practice, data on TMB and MMR genes mutational status could help in case of indeterminate MSI sensor scores or low-quality/purity tumor tissues [171,172].
About 40-50% of PCs are hereditary, frequently showing earlier onset, familial clustering, and multifocality; mutations and polymorphisms of several tumor suppressor genes and proto-oncogenes play a key role in PC onset and progression [10,169]. The impairment of MMR genes has been linked not only to sporadic PC cases (usually harboring MSH2 or MSH6 gene defects), but also to some hereditary forms of PC [4,10,169,178,179]. Lynch syndrome is an autosomal-dominant genetic disorder driven by germline mutations in MMR genes (such as MLH1, MSH2, MSH6, and PMS2), harboring greater cancer risk (especially for colorectal adenocarcinoma, but also for PC) through MSI [4,10,169,178,179]. NGS may identify germline MMR mutations, and genetic counselling for Lynch syndrome is advised for MSI-H/dMMR patients by the NCCN guidelines [4]. Unfortunately, the scant available information did not allow us to understand how many cases really harbored a Lynch syndrome and to discuss the potential differences in PD-L1 expression between somatic vs. germline MMR genes mutations.
NGS may also represent an efficient strategy to identify PC patients who may benefit from anti-PD-1/PD-L1 therapies, especially in cases with higher TMB (often associated with MMR genes alterations) [4,30,61,176]. In various neoplasms, higher TMB seems to predict favorable outcomes related to anti-PD-1/PD-L1 immunotherapy administration; indeed, the 2021 NCCN guidelines have also allowed pembrolizumab (as a second-line therapy or beyond) for mCRPCs, showing TMB > 10 mutations/Mb [4]. In PC, the median PC TMB is 3.6-4 mutations/Mb, while only 2-7.7% of cases have >20 mutations/Mb [30,61,177,180]. CRPCs usually show low TMB with alterations in key regulatory pathways (such as PI3K and androgen pathways), while ≈50% of CRPCs carry gene fusions correlating with poor prognosis: fusion-positive PCs may still harbor many neoantigens from gene fusions, potentially serving as immunotherapy targets [30,181,182]. In a PC series [30], high tumor fusion burden (TFB)-measuring the number of gene fusions in a tumor-inversely correlated with TMB and immune suppressive signatures; conversely, it was positively associated with immune infiltration, PD-L1 expression on immune cells, and immune signatures (representing activation of T cells and M1 macrophages, checkpoint inhibitors, IFN-γ-induced T-cell activity, T-effector activation, and class I antigen processing), cell cycle progression, AR signaling, and ERG/ETS transcriptional activity. Cases with ERG fusion had significantly higher overall fusion burden (p < 0.0002) and higher ERG transcriptional activity (p < 0.0001) [30]. On the other hand, Chen et al. reported that PD-L1, CD8A, or CYT expression was not associated with TMB and neoantigen number in PC [76].

PD-L1 and BRCA1/2
BRCA1 regulates the cellular DNA damage response and repair, transcriptional regulation, and chromatin modeling, while BRCA2 plays a role in the DDR processes [9,19,23,53,56,92,163]. In PC, BRCA1 co-regulates the AR activity mediating tumor progression, while BRCA2 limits the metastatic potential of PC by MMP9 downregulation and inhibition of the PI3K/AKT and MAP/ERK pathways (which also regulate PD-L1 expression) [163,164,183,184].
Loss of BRCA1 or BRCA2 is linked to a deficiency in HRR of DNA double-strand breaks; the failure of the BRCA-mediated HRR mechanism is due to combined germline and somatic mutations, favoring the activation of alternative and less effective DNA repair pathways (such as base/nucleotide excision repair or mismatch repair system) ( Figure 2) [7][8][9]19,23,53,56,92,163,164]. and inhibition of the PI3K/AKT and MAP/ERK pathways (which also regulate PD-L1 expression) [163,164,183,184].
BRCA1/2 mutations also affect the response to treatment; metastatic PC patients with germline BRCA2 mutations became resistant to the androgen deprivation therapy faster than non-carriers (13.2 vs. 28 months), showing a halved CSS [9,19,23,53,56,92,188]. Occasional studies investigated if the BRCA/DDR genes status may also affect the response to immunotherapy [19,23,92,170]. Petrylak et al. reported that most CRPC patients with DDR alterations did not respond to atezolizumab (anti-PD-L1): the DDR status was not a strong predictor of clinical benefit [19]. However, in other studies, PD-1 inhibition revealed activity in mCRPCs, including tumors lacking MSI, DDR defects, or PD-L1 expression [23,92]. In the phase II KEYNOTE-199 study, pembrolizumab (anti-PD-1) showed antitumor activity and disease control with acceptable safety in docetaxel-refractory mCRPCs, regardless of PD-L1 status, in both RECIST-measurable and non-measurable cases; 19/153 (12%) tested PCs had BRCA1/2 or ATM aberrations and higher responses rates, but it was unclear if there was a correlation between the BRCA and PD-L1 status [170]. Further studies are required.
Exceptionally, some authors performed a combined analysis of the effects of anti-PD-1/PD-L1 and PARPi in PC patients [56]. Durvalumab is a human anti-PD-L1 IgG1-K monoclonal antibody approved by the FDA for locally advanced or metastatic urothelial cancer and locally advanced, unresectable, stage 3 non-small cell lung cancer; Karzai et al. [56] found that olaparib (PARPi) plus durvalumab demonstrated activity in PC patients without biallelic inactivation of DDR pathways, reaching deep responses in mutated cases: this drug combination revealed acceptable toxicity and efficacy. It was unclear which cases were evaluated for PD-L1. Further studies are required.
Genotoxic stress and stalled DNA replication forks favor the expression of ligands for the NKG2D receptor of NK cells [195]; DDR inhibitors may also increase the NK killing activity [129]. In PC cell lines, olaparib significantly increased tumor cell sensitivity to NKmediated killing and antibody-dependent cytotoxicity (ADCC), regardless of BRCA status, PD-L1, or epithelial growth factor receptor (EGFR) modulation [129]. PARPi activates the "stimulator of interferon genes" (STING) pathway in breast carcinoma, upregulating PD-L1; in BRCA wild-type PC cell lines, STING upregulation occurred after PARPi administration without increasing the PD-L1 expression [129]. Conversely, STING was not expressed in BRCA mutant 22RV1 DU145 PC cell lines, either before or after olaparib treatment; disparities may be due to differences in olaparib exposure [129].
Olaparib may enhance the killing activity of endogenous or engineered high-affinity NK cells [107,129]. ADCC is elicited by the interaction between CD16 (FcγRIII) on NK cells and the Fc portion of IgG1 antibodies of target cells; the NK-mediated immune surveillance and killing of non-self cells are induced by target cell surface death receptors, activating a caspase cascade and resulting in apoptosis [107,129]. In PC cell lines, olaparib upregulated TRAIL-R2 (a death receptor targeted by the TRAIL ligand on NK cells), activating the caspase cascade [129]. The combined use of NK-and ADCC-mediating agents with PARPi in BRCA mutant and wild-type PC may improve treatment efficacy. A lack of PD-L1 upregulation suggests that ADCC-mediating antibodies not targeting PD-L1 (such as cetuximab) may also be exploited in combination with PARPi [107,129].
PTEN is a tumor suppressor gene encoding the phosphatase and tensin homolog, a lipid and protein enzyme. It regulates cell cycle, proliferation, and apoptosis through different pathways, including the PI3K/AKT/mTOR (which upregulates PD-L1 in PC) [10].
PTEN loss and the associated PI3K gene activation correlated to PD-L1 expression in tumors such as glioblastoma [201][202][203]. PTEN activity may be also reduced in PC; its expression can lack in whole tumor or in some areas [10,19,38,53,77,86,96]. PTEN loss is a relatively frequent, late event in mCRPCs, which is probably associated with worse prognosis [197,204,205]; however, it may indicate a sensitivity to Akt-inhibitors (such as ipatasertib) [200,206].
According to our review, 179/326 (55%) PCs showed PTEN loss by IHC: 13/137 (10%) of PTEN-cases were PD-L1+, while 10/29 (35%) of PD-L1+ cases were PTEN- [77,86,96]. The few studies investigating PD-L1 and PTEN IHC expression did not find significant correlations [38,77]; however, various (sometimes unclear) assays and scoring systems of PTEN and PD-L1 were used, and few cases were tested. PTEN loss may increase the levels of several immunosuppressive cytokines, as well as the infiltration of granulocytic myeloid-derived suppressor cells, and the inhibition of T-cell-mediated tumor killing; it can also decrease the T-cell trafficking into the tumor microenvironment [107].
The PI3K/AKT/mTOR pathway contributes to regulate PD-L1 expression in PCs [8,. Activating PIK3CA mutations increased PD-L1 expression in PC, while RAS/MAPK activating mutations may represent a "second hit" to the PTEN/PI3K/AKT pathway loss in mCRPCs [202,203]. To our review, PTEN upregulation with consequent inhibition of mTOR and PD-L1 expression has been documented in mice injected with PC cells treated with recombinant human chemerin (chemoattractant protein and PTEN activator) [107]. The chemerin-induced AKT-mTOR and PD-L1 downregulation significantly reduced PC growth: CMKLR1 knockdown abrogated this pathway. These data revealed a potential CMKLR1/PTEN/PD-L1 signaling cascade that may occur through the PI3K/AKT/mTOR pathway (as also suggested by experiments with targeted inhibitors) [105,107]. In a study [107], the PC cells of a patient also expressed CMKLR1: as in PC cell lines, chemerin may act through CMKLR1 on tumor cells to modulate PTEN and PD-L1. The type of PTEN loss may dictate the relevance of chemerin modulation in humans [107]. In PC cell lines with complete allelic PTEN loss, modulating tumor chemerin levels did not result in changes in PD-L1 expression by the tumor; however, chemerin may recruit immune effector cells into the tumor microenvironment, still having benefits in outcomes [107]. Conversely, in PCs with intact (but decreased) PTEN expression, chemerin modulation may decrease PD-L1, suppressing tumor growth and improving responses to immunotherapy [107].
Unfortunately, the relationship of the innate immune resistance (whereby PD-L1 is constitutively upregulated when PTEN is lost) has not been explored in depth in PC patients. In a few series, PD-L1 expression was independent from PTEN status [38,77,96] or PI3K pathway activation [96]. Further larger studies on human patients have to investigate the potential correlation between PTEN and PD-L1.

Comments on Some Other Genes Involved in PC Carcinogenesis and Progression
TP53 is a tumor-suppressor gene encoding a nuclear transcription factor (p53). In case of DNA damages, p53 blocks cell cycle progression, regulating the G1/S and G2/M checkpoints [42,207]. TP53 gene mutations can adversely affect PC prognosis; they seem more common in advanced stage, metastatic, and/or androgen-independent PCs [42,207]. The TP53 gene encodes 12 isoforms through alternative promoters, translation start sites, and RNA splicing. The ∆133p53 isoform has pro-tumorigenic functions, favoring cell cycle progression, anti-apoptotic activity, angiogenesis, migration, increased DNA repair and telomerase activity, and reduced chemosensitivity; it may also promote inflammation in cancer [42]. In a study [42], increased expression of the ∆133TP53β isoform seemed to define high-risk PC patients. Other authors [208] reported that only the number of active mast cells showed significant differences between low-and high-risk prognostic groups of TP53-mutated PCs [208].
The tumor-suppressor RB gene encodes a nuclear transcription factor regulating the cell cycle at the G0/G1 phase, DNA damage response, checkpoint activation, and differentiation [10,55]. At the end of the mitotic phase, the RB protein is dephosphorylated; in this active form, it interacts with E2F transcription factors and inhibits the G1/S phase transition [55]. Upon mitogen stimulation or in the late G1 phase, cyclin-dependent kinases (CDKs) phosphorylate and inhibit RB, resulting in E2F factors release from RB sequestration and allowing progression to the S phase [55]. RB may also have E2F-independent tumor-suppressor functions [209]. RB mutations allow cell cycle progression; it is controversial if the RB/p16 pathway loss is an early or late event in PC [10]. Experimental studies suggested that RB deletion could be associated with PD-L1 overexpression in mCR-PCs [55]. RB binds to the NFκB protein: in PC cell lines, CDK4/6-phosphorylated RB may promote cancer immunity through inhibition of NF-kB transcriptional activity and of PD-L1 expression [55]. CDK4/6-inhibitors, RB deletion, and irradiation can induce PD-L1 upregulation, thus causing immune evasion of PC cells [55]. A small bioactive RB-derived serine-249/threonine-252 phosphorylation-mimetic peptide decreased PD-L1 expression via NF-kB inhibition and by enhancing the anti-cancer efficacy of radiotherapy; it blocked the p65 binding to the cognate DNA sequence in the PD-L1 promoter [55]. Upon stimulation of proinflammatory cytokines, the MAP3K7-IKK signaling axis activates the transcription factor NF-kB, which regulates pro-survival genes and PD-L1 mRNA expression in various cancer types (including PC), probably favoring cancer immune escape [55].
c-MYC regulates cell growth and proliferation, cycle progression, transcription, differentiation, and apoptosis. c-MYC amplification leads to high-grade, metastatic, and/or androgen-resistant PCs [10]. However, c-MYC activation may also appear in early phases of PC [210]. In PC experimental studies, c-MYC expression and/or the combined deletion of PTEN/SMAD4 or PTEN/TP53 expanded immunosuppressive tumor-associated macrophages and myeloid-derived suppressor cells, promoting tumor immunotolerance and vascularization [132]. Moreover, c-MYC seems involved in PD-L1 regulation. The MYC inhibitor 361 (MYCi361) suppressed the tumor growth of MycCaP PC cell lines in mice, increased the tumor immune cell infiltration, upregulated PD-L1 on tumor cells, and sensitized cancer to anti-PD-1 immunotherapy [211]. In PC cell lines, the IFN-γ-induced PD-L1 mRNA and protein levels were significantly abrogated by knockdown of the histone methylation regulators WDR5 or MLL1 (not by C-MYC silencing); WDR5 seemed important for PD-L1 transcription, while OICR-9429 (antagonist of WDR5 interactions with MLL1, C-MYC, and other partners) blocked this process [10]. OICR-9429 and WDR5 knockdown also significantly reduced c-Myc recruitment [10,12].
The ERG gene encodes for a transcription factor of the ETS family; it is fused with the prostate-specific and androgen-responsive TMPRSS2 (transmembrane protease, serine 2) gene in about 50% of PCs, resulting in ERG overexpression. Two other ERG gene fusions (SLC45A3:ERG, NDRG1:ERG) can increase ERG expression, despite them occuring in <5% of PCs [166]. The ETS-TMPRSS2 fusions are apparently mutually exclusive from certain genomic aberrations: RAF-RAS-FGFR gene fusions may occur only in ETSnegative tumors [166,212]. ERG overexpression is a driver event in the transition from prostatic intraepithelial neoplasia to carcinoma [212]. Moreover, the high expression of ERG seemed associated with PCs showing advanced stage, high Gleason score, metastatic behavior, and/or shorter survival [213]. Androgen-driven ERG-TMPRSS2 gene fusions were associated with disease recurrence and tumor relapse [212,213]. Although ERG fusions/overexpression may have a diagnostic and predictive role for aggressiveness in PCs, few studies investigated their relationship with the PD-L1 status. Some authors found no significant association between PD-L1+ tumors and ERG status [38], while others reported a concordant ERG status with a nodular pattern of PD-L1 IHC positivity [77]. A trend of PD-L1+ PCs toward a lower rate of ERG positivity and higher AR expression was suggested [77]. Further data are required. The bromodomain and extraterminal (BET) family of proteins are transcriptional coactivators of cell cycle, apoptosis, migration, and invasion, frequently enhancing the expression/transcription of oncogenic drivers, such as AR and ERG in PC [213]. In PC cells lines, ERG negatively regulates miR-200c expression [86]. In a large series, miR-200a-c positively correlated to PD-L1 mRNA levels, being inversely associated with methylation of the PD-L1 promoter [86].
SPOP mutations occur in about 10-15% PCs, representing potential predictors of CRPC response to ABT [163]. These mutations are largely clustered within the MATH domain, which is responsible for substrate recognition and interaction, while the C-terminal BTB domain binds CUL3, forming the functional E3 ubiquitin ligase complex. SPOP mutations have a dominant-negative effect on substrate binding and degradation, being mutually exclusive with ERG gene fusions (the most frequent genetic alterations in PC); they increase the mRNA and protein levels of ERG and its downstream targets, promoting cellular migration and invasion [2]. Both the MATH and BTB domains are required for the SPOPmediated BRD4 ubiquitination and degradation [2,214]. In PCs, SPOP mutations confer resistance to BET inhibitors through the stabilization of BRD4. Mutations at the E3 ligase (SPOP) or the substrate (BRD4) may inhibit the SPOP-mediated BRD4 destruction by disrupting the SPOP-BRD4 interaction, stabilizing BRD4, and leading to its cooperation with AR, ERG, and other oncogenic transcription factors [214]. Cyclin D-CDK4 kinase destabilizes PD-L1 via culliculin 3-SPOP to control tumor surveillance: proteasome or ubiquitin E3 ligase inhibitors incremented PD-L1 expression [75]. Cancer-derived SPOP mutants failed to promote PD-L1 degradation by poly-ubiquitination because of their deficiency in binding to PD-L1 [75]. In human tumors, SPOP mutations in the PD-L1 C-tail are mutually exclusive with those in the substrate-interacting MATH domain [75]. SPOP can promote ubiquitin-mediated PD-L1 degradation: SPOP-mutant PCs showed increased PD-L1 expression in a series [75]. Finally, the immune-suppressed PC group showed fewer SPOP mutations than the immune-activated and non-immune class in another study (5.6%, 13.5%, and 13.2%, respectively, p < 0.001) [21].
Systematic literature reviews and metanalyses (SLRs) conducted according to the PRISMA guidelines (including an evidence-based minimum set of items for reporting) (http://www.prisma-statement.org/, accessed on 8 May 2021) are increasingly important in health care, keeping medical doctors up to date, and also representing the background for developing clinical guidelines/trials, as well as the justification for financial supports of research projects. Usually conducted by multidisciplinary teams, SLRs performed according to these guidelines could be applicable in various topics/contexts, improving the research quality not only of pure meta-analyses but also of SLRs applied to case report/series . To better present the data of our SLR, we have split the discussion of our results into different articles, highlighting relevant subtopics: PD-L1 IHC expression in PC with discussion of pre-analytical and interpretation variables; correlations of PD-L1 expression with clinic-pathological features in PC patients; PD-L1 intracellular signaling pathways in PC cells and regulation of the tumor microenvironment; pre-clinical models (cell lines, mouse models) and experimental treatments affecting PD-L1 expression in PC cells; genetic and epigenetic regulation of PD-L1; PD-L1 expression in liquid biopsies, etc. [260][261][262][263][264][265].

Conclusions
The NCCN guidelines allow pembrolizumab in selected MSI-H/dMMR mCRPC patients. Excluding cases with clear selection biases, we found 74/677 (11%) PCs with dMMR/MSI: 8/67 (12%) MSI/dMMR cases resulted in PD-L1 positivity. dMMR-PCs (tested by IHC) included ductal PCs (3%), and acinar PCs (14%), while all the cases tested for MSI by molecular analysis were acinar PCs. dMMR may be associated with worse clinical outcome, but further data are required, also as regards the differences in response to therapy.
BRCA1/2 loss of function is linked to hereditary PCs and HRR deficiency: PARPi can be administered to BRCA-mutated PC patients. In total, 15/39 (39%) PCs harbored BRCA1/2 aberrations. In a recent trial, somatic mutations in BRCA1/2 or ATM had higher responses rates after pembrolizumab in docetaxel-refractory mCRPCs. However, limited data are available for PD-L1 IHC expression in BRCA1/2-mutated PC patients; the relation of PD-L1 expression with the DDR genes status is still unclear. Further research has to verify how the efficacy of PD-1 inhibition in mCRPCs could be related to dMMR/MSI, DDR genes defects, or PD-L1 status.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/biomedicines10020236/s1, Table S1: Detailed results of the studies evaluating PD-L1 expression and microsatellite instability/mismatch repair system proteins status in patients with prostatic carcinoma.